Crossmatch testing is part of the pre-transfusion compatibility procedures performed in blood transfusion laboratories: the tests include ABO and RhD grouping, antibody screening, antibody identification, direct or indirect crossmatching.
It is routine for transfusion service laboratories to perform ABO and RhD blood typing, reverse ABO typing, and irregular antibody screening on all patients. This is performed in order to provide blood which is ABO and RhD compatible and lacking the blood group antigen to which the patient has an antibody; this will prevent or reduce the risk of transfused donor erythrocytes being destroyed by the patient. In the event of a positive antibody screen, an antibody identification investigation will be performed to identify the antibody present—often called an irregular blood group antibody as its presence is not expected, unlike regular blood group antibodies such as anti-A or anti-B, formed without known antigen stimulation and due to environmental factors. An antibody screen or identification involves the use of the patient's plasma/serum tested against a panel of specially selected erythrocyte samples, which carry the clinically significant blood group antigens against which irregular blood group antibodies are directed.
If an antibody screen returns a negative result, computer matching may be used to select suitable units for transfusion to the patient. This can occur only where appropriate validation and adherence to guidelines gives the laboratory the authorisation to do so. If however, the antibody screen is positive and an antibody is present, further compatibility tests must be carried out. This will include full antibody identification investigation and crossmatching. Compatibility tests, here referred to as crossmatching, include testing of the patient's plasma/serum against potential donor erythrocyte units.
Donor erythrocyte units should be selected as the same ABO and Rh group as the patient, and negative for the blood group antigen to which the patient has an antibody. Often this will involve blood typing of donor erythrocyte units to find those which are negative for the antigen in question, although depending on the routine testing regime of the transfusion service this testing may have been performed at the time of donor unit testing.
Conventionally, compatibility testing has been carried out as an agglutination test in a test tube. More recently this test has also been carried out using solid-phase microplate and column agglutination technologies (aka Gel, CAT). The test however, is still somewhat cumbersome requiring multiple wash steps and centrifugation. The test follows the principles of indirect antiglobulin testing (IAT, IAGT); an erythrocyte suspension is allowed to incubate with a sample of plasma/serum/blood typing reagent or control. During this first step, if an antibody is present and the antigen to which it is specific is also present on the erythrocytes, binding of antibody to antigen will occur—this step is called sensitisation. Following sensitisation, wash steps are required to separate unbound antibody in solution, from the sensitised erythrocytes. Following this wash step, an anti-human globulin reagent is added; this contains anti-human IgG antibodies and often anti-C3. If antibody has sensitised the erythrocytes the anti-human IgG will bind to the IgG antibody on the erythrocytes causing haemagglutination. In conventional testing, IgM incompatibility is identified through direct haemagglutination, without the sensitisation pre-step. Haemagglutination is the end point used for detection of a sensitisation reaction. Failure to remove unbound IgG can lead to neutralisation of the anti-human IgG present in the anti-human globulin reagent and potentially lead to a false negative result. This is usually controlled by the addition of IgG sensitised cells to all negative IAT tests; here a positive result shows that the anti-human IgG is available and has not been neutralised, and therefore adequate washing/removal of unbound antibody has occurred. If the test is negative it may demonstrate that the binding regions of the anti-human IgG are blocked, and most likely ‘neutralised’, and it may be concluded that the test is invalid due to insufficient washing or unbound antibody removal.
Current state-of-the-art includes commercially available systems such as Immucor Capture-R, and BioRad ID-System and Ortho Clinical Diagnostics BioVue and ID-MTS systems, and although many other variations are now available they are very similar in principle to the systems mentioned above. Solid phase systems, such as the Immucor Capture-R Select involve binding of the donor erythrocytes to a microplate well, followed by incubation with patient plasma/serum, wash steps and then finally addition of indicator cells (cells coated with anti-D and a level of anti-IgG) so where antibody has sensitised the bound donor erythrocytes the anti-IgG on the indicator cells will also bind to the donor antibody bound to the donor erythrocytes. In this example, wash steps are required to remove unbound antibody. Column agglutination techniques use a microtube containing anti-human IgG (and/or anti-C3) and use a well above the microtube to allow incubation/sensitisation of donor cells with patient plasma/serum, before centrifugation through the column containing anti-human IgG. In this case centrifugation is used to separate the unbound antibody, as the erythrocytes are forced through the microtube by centrifugation leaving unbound antibody in the well above the microtube as the centrifugal force is appropriate to force cells down but will not be sufficient to allow unbound antibody into the anti-IgG column.
Current systems as described above, use either centrifugation and/or wash steps to separate the sensitised erythrocytes from unbound antibody. Such procedures are time and reagent consuming and thus render prior art processes less suitable for rapid high-throughput screening.
The presence of antigens (including blood group antigens) on the surface of erythrocytes forms the basis of many immunological tests including, for example blood typing assays which use non-agglutination protein microarrays, in which an immobilised antibody binds to an antigen on the surface of the erythrocytes, and the presence of erythrocytes so immobilised is detected (J S Robb et al 2006). Antibody microarray technology can also be used to phenotype erythrocytes by detecting complex mixtures of antigens on cell surfaces (C J Campbell et al 2006). The antigens expressed by erythrocytes are both sugar antigens, which tend to be well presented and easily accessible, and protein peptide antigens, which are epitopes of transmembrane proteins and therefore buried and held more closely to the cell surface, and these were successfully differentiated using the correct choice of antibodies.